Comparative Study of Sonodynamic and Photoactivated Cancer Therapies with Re(I)-Tricarbonyl Complexes Comprising Phenanthroline Ligands.

{"full_text": " pubs.acs.org/jmc Article\n\n\n\n Comparative Study of Sonodynamic and Photoactivated Cancer\n Therapies with Re(I)-Tricarbonyl Complexes Comprising\n Phenanthroline Ligands\n Rajesh Kushwaha, Virendra Singh, Silda Peters, Ashish Kumar Yadav, Tumpa Sadhukhan,*\n Biplob Koch,* and Samya Banerjee*\n Cite This: J. Med. Chem. 2024, 67, 6537\u22126548 Read Online\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 11:46:07 (UTC).\n\n\n\n\n ABSTRACT: Herein, we have compared the effectivity of light-\n based photoactivated cancer therapy and ultrasound-based\n sonodynamic therapy with Re(I)-tricarbonyl complexes (Re1\u2212\n Re3) against cancer cells. The observed photophysical and TD-\n DFT calculations indicated the potential of Re1\u2212Re3 to act as\n good anticancer agents under visible light/ultrasound exposure.\n Re1 did not display any dark- or light- or ultrasound-triggered\n anticancer activity. However, Re2 and Re3 displayed concen-\n tration-dependent anticancer activity upon light and ultrasound\n exposure. Interestingly, Re3 produced 1O2 and OH\u2022 on light/\n ultrasound exposure. Moreover, Re3 induced NADH photo-\n oxidation in PBS and produced H2O2. To the best of our\n knowledge, NADH photo-oxidation has been achieved here with\n the Re(I) complex for the first time in PBS. Additionally, Re3 released CO upon light/ultrasound exposure. The cell death\n mechanism revealed that Re3 produced an apoptotic cell death response in HeLa cells via ROS generation. Interestingly, Re3\n showed slightly better anticancer activity under light exposure compared to ultrasound exposure.\n\n\n \u25a0 INTRODUCTION\n The widespread use of platinum-based chemotherapeutics for\n 365 nm light-activated anticancer activity of Re(I) complexes\n via the combined effect of CO release, 1O2 generation, and\n cancer treatment is now suffering from several drawbacks, such rhenium-containing photoproducts.26 Gasser et al. have shown\n as side effects and drug resistance.1\u22123 Over the last few the ROS-mediated apoptosis/necrosis of HeLa cells induced\n decades, researchers have developed new treatment modalities by Re(I)-tricarbonyl complexes upon 350 nm light exposure.30\n with new anticancer compounds to overcome the drawbacks of In another report, the Gasser group showed cell-specific 350\n the clinical Pt(II)-based cancer drugs.4\u22128 In this regard, nm light-triggered anticancer activity via 1O2 generation-\n selective activation of a cancer drug at the cancer site is proven mediated DNA damage by a neuropeptide, bombesin,\n to be effective in reducing drug side effects.7\u22129 Based on this conjugated Re(I) tricarbonyl.31 Pan et al. have reported\n concept, cancer treatment modalities like photoactivated dinuclear phosphorescent Re(I)-tricarbonyl complexes show-\n cancer therapy (PACT) and sonodynamic therapy (SDT) ing 425 nm light-induced phototoxicity by lysosomal\n were developed.10\u221213 Metal complexes showed positive membrane permeabilization and ROS production.28 Maisuls\n response as the cancer prodrugs in PACT and SDT.14\u221217 et al. have reported a dppz-Re(I)-tricarbonyl complex showing\n Several 3d\u22125d metal (such as VO(II), Zn(II), Ru(II), Ir(III), 365 nm light-triggered cancer cell death via ROS-induced\n Pt(IV), and Os(II), etc.) complexes presented light-triggered DNA damage.32 The Meggers group has explored the potential\n anticancer activities through different modes of action.18\u221223 of Re(I) tricarbonyl to achieve visible-light anticancer\n Recently, metal complexes are also attracting attention as activity.33 Importantly, most of the reported Re(I)-tricarbon-\n sonosensitizers in SDT.24 A cyanine-based Pt(II) complex is\n reported to induce ferroptotic cancer cell death upon\n ultrasound exposure.24 Re(I)-tricarbonyl complexes have Received: December 31, 2023\n attracted notable attention as PACT agents due to their Revised: March 23, 2024\n ROS generation abilities and CO-releasing properties on light Accepted: March 29, 2024\n exposure.25\u221230 ROS generation and CO-releasing abilities of Published: April 11, 2024\n Re(I)-tricarbonyl complexes made them good dual-action\n PACT agents.26,27 Wilson and co-workers have reported the\n\n \u00a9 2024 American Chemical Society https://doi.org/10.1021/acs.jmedchem.3c02485\n 6537 J. Med. Chem. 2024, 67, 6537\u22126548\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n\n\n\nFigure 1. (a) Structure of complexes Re1\u2212Re3. (b) Absorption spectra of Re1\u2212Re3 in DMSO/H2O (10:90 v/v) solution. (c) Emission spectra of\ncomplexes Re1\u2212Re3 in DMSO/H2O (10:90 v/v) solution (20 \u03bcM, \u03bbex = 370 nm for Re1/Re3 and 390 nm for Re2).\n\nyl-based PACT agents were excited with UV light to achieve\nanticancer activities and, thus, are not highly suitable for\n \u25a0 RESULTS AND DISCUSSION\n Synthesis and Structural Analysis. Complexes Re1\u2212\nclinical applications.26\u221232 Limited report is available for high Re3 (Figure 1a) were synthesized in good yield (80\u221290%) by\nwavelength light-induced anticancer effects, but their activity reacting stoichiometric amounts of rhenium pentacarbonyl\nwas due to the presence of the porphyrin moiety, not the Re(I) chloride, [Re(CO)5Cl], and 1, 10-phenanthroline-based\ncore.34 Thus, Re(I)-tricarbonyl complexes, which can show ligands in toluene under reflux conditions.27,38\u221243 The\nvisible-light-induced anticancer activity, are needed to broaden diffusion of hexane in an acetone solution of Re2 yielded\nthe scope of Re(I) tricarbonyls in clinical PACT applications. diffraction-quality single crystals of the complex. All of the\n The low tissue penetration power of visible light (except red complexes were characterized with NMR, IR, HRMS,\nlight) compromises the overall potential of PACT.12,16 elemental analysis, and UV\u2212vis spectroscopy (Figures S1\u2212\n S12). NMR (Figures S1\u2212S6) and elemental analysis data\nRecently, SDT with ultrasound as the drug stimulus has revealed the high purity of Re1\u2212Re3. The HRMS of Re1\u2212Re3\nevolved to overcome this limitation of PACT.8,15,24,35 Of late, in CH3CN displayed m/z peaks corresponding to the [M +\nour group and Zhang\u2019s group have shown that ultrasound can Na]+ species (Figures S7\u2212S9). The FT-IR spectra of Re1\u2212\nalso trigger anticancer activities out of Re(I) tricarbonyls by Re3 showed three bands between 1880 and 2030 cm\u22121,\nreleasing CO gas and ROS generation, establishing the confirming the presence of three facial carbonyl groups\nusefulness of Re(I) tricarbonyls in SDT.36 Till today, a (Figures S10\u2212S12).27,38\u221242 The UV\u2212vis spectra of Re1\u2212Re3\ncomparative study between SDT and PACT with Re(I)- in 10% aq. DMSO displayed a band around 370\u2212390 nm.\ntricarbonyl complexes is missing. Such a study could provide These bands correspond to the Re(I)-to-ligand charge transfer,\ninformation about the SDT and PACT performances of Re(I)- which dominates in the photophysical/chemical properties of\ntricarbonyl complexes under ultrasound and light exposure, the Re(I)-tricarbonyl complexes.27,38\u221242 Complex Re3 also\n displayed an additional band around 445 nm, corresponding to\nrespectively. A comparative study could also indicate the\n ligand-based n\u2212\u03c0* transition (Figure 1b).39,40 Re1 and Re3\npossibility of multimodal cancer therapy combining SDT and (in 10% aq. DMSO) on excitation at 370 nm exhibited\nPACT. photoluminescence with the maxima near \u223c590 nm. Re2\n In this work, we have used simple Re(I)-tricarbonyl showed photoluminescence at ca. 440 nm under similar\ncomplexes, viz., [Re(phen)(CO)3Cl] (Re1), [Re(phen-NO2)- experimental conditions upon excitation at 390 nm (Figure\n(CO)3Cl] (Re2), and [Re(phen-NH2)(CO)3Cl] (Re3), where 1c). The emission properties of these complexes were used to\nphen = 1,10-phenanthroline; phen-NO2 = 5-nitro-1,10- determine their internalization inside cancer cells.\nphenanthroline; phen-NH2 = 5-amino-1,10-phenanthroline, X-ray Single-Crystal Structure. The crystal structure of\nto get visible light-assisted PACT and SDT.37\u221243 We also Re2 revealed its distorted octahedral geometry (Figure 2). Re2\ncompared the anticancer activity of these complexes in the has a P121/n1 space group and monoclinic system. The crystal\npresence of ultrasound and visible light. Important findings of structure of Re2 suggested that there were four molecules of\n Re2 within the unit cell, while only one molecule was within\nthis work comprise: (i) both light and ultrasound can activate\n the asymmetric unit (Figure S13). As depicted in Figure 2,\nthe Re(I)-tricarbonyl complexes to induce apoptosis via ROS Re(I) is coordinated with three CO, the N,N donor chelating\ngeneration and NADH oxidation. (ii) Cytotoxicity under ligand (5-nitro-1,10-phenanthroline), and chloride. The\nultrasound was a bit less than light, but ultrasound with high structure of Re2 revealed that two of the CO were positioned\ntissue penetration might be useful for further in vivo studies, at the equatorial position and one CO at the axial position,\nwhich will be the topic of our future investigation. trans to Cl. Some important bond angles and lengths are listed\n 6538 https://doi.org/10.1021/acs.jmedchem.3c02485\n J. Med. Chem. 2024, 67, 6537\u22126548\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n and LUMO was located on the substituted phen ligand (Figure\n 3a). Different vertical transition energies are given in Tables\n S3\u2212S5. The oscillator strengths for each transition, repre-\n sented in Tables S3\u2212S5, show that the transitions are allowed.\n Furthermore, we have carried out natural transition orbital\n analysis on the complexes\u2019 lowest and most intense transitions\n (Figures S16\u2212S18). For Re1 and Re2, the red-shifted band\n corresponds to the HOMO \u2192 LUMO transition indicating\n metal-to-ligand charge transfer (1MLCT). In the case of Re3,\n there was a HOMO-1 \u2192 LUMO transition with the\nFigure 2. ORTEP view of Re2 showing 50% probability thermal participation of the HOMO \u2192 LUMO transition suggesting\nellipsoids (CCDC = 2299334). a mixture of ligand-to-ligand charge transfer (1LLCT) and\n 1\n MLCT. The triplet excited states (T1) were optimized to gain\nin Table 1. Important crystal parameters and refinement details insights into spin density and SOMO plot nature and to\nof Re2 are provided in Table S1. The N1\u2212Re\u2212N2 angle is determine the energy gap between T1 and S0, \u0394EST (Figure 3c\n and Table S6). The optimized singlet and triplet structures of\nTable 1. Important Bond Lengths and Bond Angles of Re2 Re1\u2212Re3 were more or less similar (Figures S19 and S20).\n bond lengths (\u00c5) bond angles (deg) The spin density plots of Re1\u2212Re3 in their triplet state\n displayed localization around the bidentate ligand and the Re\nRe\u2212Cl 2.472(2) Cl\u2212Re\u2212 84.1(1) N2\u2212Re\u2212C3 98.1(2)\n N1 atom (Figure 3b). The SOMO plots (Figure S21) suggested an\n 3\nRe\u2212N1 2.179(4) Cl\u2212Re\u2212 81.2(1) C8\u2212N3\u2212 118.2(5) MLCT character for Re1 and Re2 and mixed\n 3\n N2 O4 MLCT\u22123LLCT in Re3, consistent with singlet ground states.\nRe\u2212N2 2.184(5) Cl\u2212Re\u2212C1 92.3(2) C8\u2212N3\u2212 117.3(5) Moreover, the energy difference \u0394EST values obtained for\n O5\nRe\u2212C1 1.928(5) Cl\u2212Re\u2212C2 92.9(2) O4\u2212N3\u2212 124.5(6) Re1\u2212Re3 range between 2 and 2.5 eV (Table S6), and these\n O5 values significantly exceeded the minimum energy required,\nRe\u2212C2 1.925(6) Cl\u2212Re\u2212C3 178.3(2) Re\u2212N1\u2212 114.6(3) 0.98 eV to transform 3O2 into 1O2 (Figure 3d).45\n C14 Intracellular Uptake and Cytotoxicity Assay. Adequate\nRe\u2212C3 1.941(6) N1\u2212Re\u2212 75.0(2) Re\u2212N2\u2212 114.9(3) intracellular uptake for any cancer therapeutic is essential for in\n N2 C15\nC8\u2212N3 1.480(8) N1\u2212Re\u2212 97.9(2) Re\u2212C1\u2212O1 178.8(6) vitro and in vivo anticancer activities.47\u221249 Henceforth, we\n C1 examined the cellular uptake of Re2 and Re3 in HeLa cells\nN3\u2212 1.219(7) N1\u2212Re\u2212 94.3(2) Re\u2212C2\u2212O2 177.6(5) after 6 h of incubation by measuring in-cell fluorescence from\n O5 C3 the complexes through flow cytometer analysis.50,51 The flow\nN3\u2212 1.218(7) N2\u2212Re\u2212 97.6(2) Re\u2212C3\u2212O3 173.9(6) cytometry data (Figure 4) clearly depicted that Re2 and Re3\n O4 C2\n internalized significantly into the HeLa cells within 6 h of\n incubation. The significant cellular uptake of the complexes\ncharacteristically small (75.0\u00b0), in line with the previously encouraged us to study the cytotoxicity of complexes against\nreported structures of similar Re(I) tricarbonyls.27,38,42 The HeLa (cervical cancer cell line), MCF-7 (breast cancer cell\nbond angles N1\u2212Re\u2212Cl (81.2\u00b0) and N2\u2212Re\u2212Cl (84.1\u00b0) were line) cancer cell line, and normal HEK (human embryonic\nbelow 90.0\u00b0, indicating that the phenanthroline moiety is little kidney) cell line in both dark and after light exposure using the\nbent toward the axial Cl (Table 1).27,38,42 The angles C1\u2212Re\u2212 MTT assay.52\u221255 Re1 did not present any significant\nCl (92.3\u00b0) and C2\u2212Re\u2212Cl (92.9\u00b0) were higher than 90\u00b0, cytotoxicity against any of the tested cell lines with the\nindicating that CO groups are bent toward the axial CO (Table presence or absence of light (400\u2212700 nm, 5 J cm\u22122) (Table\n1).27,38,42 The \u2212NO2 group on phenanthroline was not planar, 2). Earlier, the Mascharak group reported that Re1 was also\nas was evident from the torsion angles of C7\u2212C8\u2212N3\u2212O4 nontoxic against MDA-MB231 cells.38 Re2 and Re3 did not\n(\u223c144.8\u00b0) and C7\u2212C8\u2212N3\u2212O5 (\u223c35.2\u00b0). The nonplanarity\n produce any toxic response against HeLa and MCF-7 cells in\nof the \u2212NO2 group might be due to the interaction between\n the dark condition, even at 50 \u03bcM concentration. But,\nthe nitro group oxygen and the neighboring molecule\u2019s oxygen\n interestingly, Re2 and Re3 significantly decreased the cell\nof axial CO (Figure S13).\n Computational Studies. Figure 1a shows the structures of viability of both MCF-7 and HeLa cancer cells upon light\nthe Re(I) carbonyl complexes investigated in this work. Re1\u2212 exposure in a concentration-dependent manner. The com-\nRe3 were optimized in their singlet ground states (S0), first plexes Re2 and Re3 showed ca. 4 times higher anticancer\nexcited singlet state (S1), and in their triplet excited states (T1) activity in HeLa cells than in MCF-7 cells (Table 2). In\nat the \u03c9B97X-D/Def2-TZVP in DMSO.44\u221246 As seen in contrast, dinuclear Re(I)-tricarbonyl complexes reported by\nFigure 1a, all complexes investigated are very similar in Mao et al. have presented almost the same phototoxicity in\nstructure where Re(I) is attached to 3 CO groups, 1 Cl, and both HeLa (IC50 = 1.8 \u00b1 0.2 \u03bcM) and MCF-7 (IC50 = 2.2 \u00b1\none 1,10-phenanthroline substituted at 5-C. Figure S14 shows 0.3 \u03bcM) cancer cells.56 Both complexes showed photo-\nthe optimized structures, and Figure S15 and Table S2 show cytotoxicity index (ratio of dark IC50 to light IC50) value\nthe FMO plots and their energies. Figure 3a reveals that for >25 against HeLa cells, indicating their selective cancer cell-\nRe1 and Re2, the HOMO is localized over the Re, CO, and Cl, killing ability under light. More importantly, the cytotoxic\nwhereas the LUMO is predominantly located on the phen effect of Re2 and Re3 against normal HEK cells (IC50 > 50\nligand. In the case of Re3, HOMO-1 was located on the Re, \u03bcM) was lower than cancer cells with significant SI (SI =\nCO, and Cl, HOMO was delocalized on the entire molecule, selectivity index = ratio of IC50 against the normal cell to IC50\n 6539 https://doi.org/10.1021/acs.jmedchem.3c02485\n J. Med. Chem. 2024, 67, 6537\u22126548\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n\n\n\nFigure 3. (a) Difference between HOMO and LUMO of complexes Re1\u2212Re3; (b) spin density isosurface of Re1\u2212Re3 at the T1 optimized\ngeometries; (c) calculated energy level of lowest triplet energy (T1) and excited singlet (S1) levels of Re1\u2212Re3; and (d) adiabatic singlet\u2212triplet\nenergy gap (\u0394S0\u2212T1) of Re1\u2212Re3.\n d d\n\n\n\n\n sound can be useful.59,60 Ultrasound has excellent tissue-\n penetrating ability and thus can activate the drug even at the\n deep-seated tumor site.59,60 Recently, we have shown that\n ultrasound can also release CO from the Re(I)-tricarbonyl\n complex.59,60 To realize the efficacy of Re1\u2212Re3 in SDT, their\n anticancer activities were studied under ultrasound irradiation\n (1.5 W cm\u22122). Moreover, we were interested in comparing the\n effects of light and ultrasound as drug activators. Interestingly,\n Re3 showed significant anticancer activity on ultrasound\n exposure with the IC50 value ca. 5 \u03bcM against HeLa cells.\n Thus, Re3 presented a marginally higher anticancer effect in\n the presence of visible light (IC50 = ca. 2 \u03bcM) than the\n ultrasound. The low micromolar IC50s of Re3 under light and\nFigure 4. Flow cytometric analysis data showing the uptake of Re2 ultrasound indicate that this complex can be used in\nand Re3 (5 \u03bcM) in HeLa cells after incubation for 6 h of incubation. photoactivated cancer therapy as well as in cancer SDT. The\n SDT performance of Re3 is encouraging, as it can be used for\nagainst the cancer cell, SI > 25 for both Re2 and Re3), deeply buried tumor as a sonosensitizer.\nindicating their high selectivity toward cancer cells. ROS Generation. Reactive oxygen species (ROS)\n Although photoactivated cancer therapy has offered production during cellular metabolism plays an important\nsignificant therapeutic results, the low tissue penetration role in maintaining cellular redox harmony.61,62,64 But any\npower of light has compromised its effect on deep-seated artificial increment in the intracellular ROS levels results in the\ntumors.8,57\u221260 In this regard, only the red light-absorbing damage of many cellular components, such as lipids, proteins,\nphotoactive molecules can be used to treat deeply buried DNA, etc., that highly compromises regular cellular activity\ntumors.7,57,58 As our complexes do not have any absorption in and leads to cell death.61,62 ROS generation have been a\nthe red light region, so might be useful for superficial cancers.8 pivotal mechanism in PDT as well as in SDT, which holds\nTo overcome the limitation associated with the light\u2019s tissue promising potential for anticancer effects.59\u221262,64 The gen-\npenetration, the concept of SDT with ultrasound as a drug eration of ROS like 1O2 and OH\u2022 plays a vital role in cancer\nactivator was introduced recently.8,59,60 To use Re(I)- therapy due to their oxidative stress-mediated destruction of\ntricarbonyl complexes for deep-seated tumor therapy, ultra- cancer cells.61,62 The singlet oxygen production by photo/\n 6540 https://doi.org/10.1021/acs.jmedchem.3c02485\n J. Med. Chem. 2024, 67, 6537\u22126548\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\nTable 2. IC50 Values (\u03bcM), Photocytotoxicity Index (IC50 dark/IC50 light), and Selectivity Index (SI, SI = IC50 normal cell/\nIC50 light, cancer cell) of Re1\u2212Re3, Cisplatin, and Relevant Re(I)-Tricarbonyl Complexes\n complex HeLa MCF-7 HEK-293\n dark light ultrasound photocytotoxicity index SI dark light photocytotoxicity index SI dark\n Re1a >50 >50 >50 >50 - >50\n Re2a >50 2.0 \u00b1 0.4 >10 >25 >25 >50 9.2 \u00b1 0.4 >5 >5 >50\n Re3a >50 1.9 \u00b1 0.4 5.01 >25 >25 >50 8.8 \u00b1 0.5 >5 >5 >50\n Re\u2212NH2b >100 17.3 \u00b1 2.9 >6\n Re-COOHb >100 9.3 \u00b1 2.2 >10\n Re-DAPTA1c >200 5.9 \u00b1 1.4 >33.9\n Re-Cyd 1.99\n cisplatine 71.3 68.7 1.1 69.7\na\n This work, cells were incubated with the complexes for 6 h in the dark, followed by light (30 min, 400\u2212700 nm, 5 J cm\u22122) and ultrasound (10\nmin, 1.5 W cm\u22122) irradiation and a further 18 h recovery time. bFrom ref 30 (light, 365 nm). cFrom ref 26 (light, 365 nm). dFrom ref 36 (US, 0.3\nW cm\u22122). eFrom ref 50 (light, 400\u2212700 nm).\n\n\n\n\nFigure 5. (a) 1O2 generation induced by Re3 (10 \u03bcM) upon light (400\u2212700 nm, 5 J cm\u22122) and ultrasound (1.5 W cm\u22122) irradiation in DMSO/\nH2O (2:98 v/v) solution. (b) OH\u2022 generation induced by Re3 (10 \u03bcM) upon light (400\u2212700 nm, 5 J cm\u22122) and ultrasound (1.5 W cm\u22122)\nirradiation in DMSO/H2O (2:98 v/v) solution. (c) ROS generation induced by Re3 (2 \u03bcM) upon light (400\u2212700 nm, 5 J cm\u22122) and ultrasound\n(1.5 W cm\u22122) irradiation in HeLa cells after 30 min of incubation. Scale bar: 400 \u03bcm.\n\nsono-sensitizers on light/ultrasound irradiation has been Zhang\u2019s group has explored efficient singlet oxygen production\nreported to induce cancer cell killing.62,63 1O2 generation by by Ir(III)-porphyrin complexes, Pt(II) complexes, and Ru(II)\nphotosensitizers has been an important cofactor and well- polypyridyl complexes.15,17,24 In sonodynamic therapy, ultra-\nexplored cell death mechanism for anticancer activity. sound waves cause cavitation within the cellular medium and\nSimilarly, sonosensitizers have also been explored to produce form bubbles. The formed bubbles further burst violently and\n1 produce a high energy. The produced energy activates the\n O2 upon ultrasound exposure. For example, Zhu et al.\nreported a smart sonosensitizer PpIX@HMONs-MnOx-RGD sonosensitizer, which then most likely transfers the energy to\n(Arg-Gly-Asp) (named PMR) to produce 1O2 by ultrasound\n 3\n O2 to generate 1O2.15,17,36 As the TD-DFT calculation\nirradiation.63a Similarly, Cheng and co-workers have shown depicted that Re1\u2212Re3 possesses sufficient energy difference\n1\n O2 generation by HXV2O5 nanocatalysts.63b Ultrasound has \u0394EST (\u0394S0\u2212T1) to generate 1O2 from 3O2 (Figure 3d), the 1O2\n d d\n\n\n\n\nalso been reported to enhance the 1O2 generation efficiency of generation ability of Re2 and Re3 was studied using the 9,10-\nphotosensitizers. For example, Karanl\u0131k et al. have shown an diphenyl anthracene (DPA) probe.36 In the dark or in the\nincrease in 1O2 quantum yield by 60% upon ultrasound absence of ultrasound, the absorption intensity of the DPA-\nirradiation than light irradiation.63c Several metal complexes based band was not changed notably in the presence of Re2/\nhave also been reported to generate 1O2 under ultrasound Re3, indicating that these complexes are unable to produce a\nexposure. Recently, Xu et al. have shown the high 1O2 significant amount of 1O2 in the absence of any drug stimulus.\ngeneration efficiency by Ru(II)-based sonosensitizers.63d The 1O2 generation efficiency of Re2/Re3 was incredibly\n 6541 https://doi.org/10.1021/acs.jmedchem.3c02485\n J. Med. Chem. 2024, 67, 6537\u22126548\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n\n\n\nFigure 6. (a) Light-triggered NADH oxidation by Re3 (10 \u03bcM) in DMSO-PBS (2:98 v/v) solution. (b) H2O2 generation by Re3 (10 \u03bcM) in the\npresence of NADH upon light exposure.\n\nincreased after exposure to light (400\u2212700 nm, 5 J cm\u22122) and Oxidation of NADH to NAD+. In addition to serving as a\nultrasound (1.5 W cm\u22122), as was observed from the significant crucial coenzyme in more than 400 oxidoreductases in live\ndecrease in the absorption intensity of the DPA-based band at cells,65,66 NADH is recognized as the main e\u2212 source in the\n360\u2212410 nm (Figure S22). Interestingly, Re3 produced a mitochondrial electron transport chain (ETC).65\u221272 Moreover,\nhigher amount of 1O2 under light exposure (Figure 5a). The the overall in-cell NADH level is higher in cancerous cells than\n1\n O2 quantum yield of these complexes was further calculated in normal cells to ensure high cell proliferation.69,73 Hence-\ntaking [Ru(bpy)3]Cl2 as a standard (\u03a6\u0394R = 0.22) in aq. forth, any artificial depletion in NADH in cancer cells can\nDMSO.17 The result revealed that the 1O2 quantum yield for provide a multitargeting anticancer activity.65\u221272 Recently, the\nRe3 (\u03a6\u0394Re3 = 0.24) was higher than those for Re2 (\u03a6\u0394Re2 = Sadler group, Huang group, and our group have successfully\n0.16) and Re1 (\u03a6\u0394Re1 = 0.05) (Figure S23). This result demonstrated that in-cell artificial oxidation of NADH/\nindicated that Re3 might be a better type-II PDT agent than NAD(P)H by metal complexes leads to cell death.65\u221272\nRe1 and Re2. On ultrasound exposure, the rate of DPA Motivated by the appealing excited-state photochemistry and\noxidation for 1O2 generation was quantified by measuring the ROS generation ability of Re3, we investigated the light-\ndecrease in the time-dependent DPA-based peak (ca. 378 nm) triggered oxidation of NADH to NAD+. The gradual decrease\nabsorbance. The DPA oxidation rate constant was observed to in the intensity of the NADH-based band at ca. 339 nm and\nbe 0.0068 min\u22121 for Re3, which was higher than those for Re2 subsequent increase in the intensity of NAD+ based at ca. 260\n(0.0049 min\u22121) and Re1 (0.0011 min\u22121). This observation is nm indicated the oxidation of NADH to NAD+ (Figure\naligned with the photo/sonotoxicity presented by Re2/Re3 6a).65\u221272 It was found that Re3 (TON = 4.9, TOF = 16.3 h\u22121)\nand can be attributed to the higher light absorption of Re3. significantly oxidizes NADH to NAD+ under the influence of\n visible light (400\u2212700 nm, 5 J cm\u22122) (Figure 6a) and does not\nFurthermore, we examined the OH\u2022 generation ability using\n exhibit any effect without light (Figure S29). NADH oxidizing\nthe OH\u2022 probe, methylene blue (MB).36 Following a trend\n ability of Re3 on light irradiation was supported by the excited-\nsimilar to 1O2 production, no significant OH\u2022 generation by\n state redox potential calculation. At the excited state, the\nRe2/Re3 was observed under dark or without ultrasound\n reduction potential of Re3 ([Re3*]0/[Re3]\u2212 = +0.83 V)\nirradiation (Figure S24), and OH\u2022 generation was increased (Table S7) was higher than NADH ([NAD]+/[NADH] =\nafter visible light or ultrasound exposure (Figure S25). Re3 +0.32 V).66 Therefore, NADH transferred electrons to Re3*\nagain produced more OH\u2022 compared to Re2 (Figure 5b). and was converted into NADH\u2022+, which further converted O2\nThus, these complexes show anticancer activity under light via to H2O2. H2O2 production was detected using peroxide test\nboth type-I and type-II PDT pathways. It is important to strips (Figure 6b). A similar mechanism is also reported with\nmention that, although several Re(I)-tricarbonyl complexes are Ir(III) complexes.65\u221268 Overall, the above results indicated\nreported to produce 1O2 upon light or ultrasound expo- that the photocytotoxicity of Re3 could be a combined effect\nsure,36,59 OH\u2022-generating Re(I)-tricarbonyl complexes are not of type-I and type-II ROS generation mechanisms and in-cell\nreported yet. NADH oxidation. It is important to mention here that the\n Furthermore, the ROS generation ability of Re2 and Re3 in reported metal complexes showing NADH oxidation are either\nthe HeLa cells was visualized using the fluorescent probe, Ru(II) or Ir(III) complexes, and this is the first Re(I)-\nDCFDA (2,7-dichlorodihydrofluorescein diacetate), and fluo- tricarbonyl complex showing NADH oxidation.\nrescence microscopy.61\u221263 In this approach, the generated CO Release. Several reports suggested that Re(I)-\nROS is known to transform the nonfluorescent DCFDA into tricarbonyl complexes can release CO upon light or ultrasound\nthe highly fluorescent 2\u2032,7\u2032-dichlorofluorescein (DCF).61\u221263 irradiation.26,27,36\u221238 For example, Wilson et al. and Mascharak\nComplexes Re2 and Re3 showed significant ROS generation at et al. have established that Re(I) tricarbonyls can serve as CO-\ntheir respective IC50 concentrations only upon light and releasing molecules under the influence of light.26,27,38 The\nultrasound exposure (Figures 5c and S26\u2212S28). Importantly, Wilson group recently reported the enhanced cytotoxicity of\nin the absence of drug stimuli (light or ultrasound), Re2 and Re(I) tricarbonyls due to CO release in photoactivated cancer\nRe3 were unable to generate observable ROS (Figure S26), therapy.26 Furthermore, Zhang et al. and our group proved\nindicating that the Re2/Re3 + light/ultrasound combination that CO can be released from Re(I)-tricarbonyl complexes\nconsiderably enhanced ROS generation (DCF green fluo- with ultrasound exposure to enhance anticancer activities.36,59\nrescence) in HeLa cells and promoted cell death via oxidative Therefore, the release of CO from Re3 was investigated by a\nstress. UV\u2212vis study under the influence of visible light and\n 6542 https://doi.org/10.1021/acs.jmedchem.3c02485\n J. Med. Chem. 2024, 67, 6537\u22126548\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n\n\n\nFigure 7. Change in UV\u2212vis spectra reflecting CO release from Re3 (15 \u03bcM) in DMSO-PBS (2:98 v/v) solution (pH 7.4) over a period of 60 min\nunder (a) visible-light irradiation and (b) ultrasound irradiation.\n\n\n\n\nFigure 8. Light/ultrasound-triggered apoptotic cell death of HeLa cells induced by Re3 (2 \u03bcM) probed by Hoechst and PI. Scale bar: 100 \u03bcm.\n\nultrasound. The change in UV\u2212vis absorption spectra taken in and gives an estimate of the reaction barrier. The irradiation of\nPBS buffer (pH 7.4) with time indicated the loss of CO with light or ultrasound provided this amount of energy.\nlight exposure (Figure 7a). The presence of well-defined Apoptosis Study. The increment in cytotoxicity and ROS\nisosbestic points in the UV\u2212vis spectra of Re3 indicated the generation ability of Re2 and Re3 after light/ultrasound\nCO release upon visible-light exposure.26,27,38 CO release from irradiation prompt further investigation into the cell death\nRe1 was reported by the Mascharak group with high-energy mechanism caused by the (Re2/Re3) + light/ultrasound\nlight (<315 nm).38 Possessing an absorption maximum similar combination against HeLa cells. Hoechst/PI dual staining was\nto that of Re1, CO release from Re2 might be achieved with used to determine the cell death mechanism.63,74,75 Hoechst\nhigher energy UV light. A similar observation was also found 33342 dye is known to probe the nuclei of both apoptotic and\nwith the change in UV\u2212vis spectra under the influence of live cells as it produces light-blue emission with live cells\u2019\nultrasound (Figure 7b), indicating that Re3 can release CO nuclei and strong blue emission upon binding with the nuclei\nunder the influence of light and ultrasound. Furthermore, this of apoptotic cells.74 The other dye, PI (propidium iodide),\nresult was also complimented by IR spectroscopy, where the stains the nuclei of dead or dying cells and produces bright-red\nintensity of the Re3-based IR peaks between 1880 and 1920 fluorescence.75 As shown in Figure 8, after Re2/Re3 (at their\ncm\u22121 corresponding to the CO stretching frequency decreased respective IC50 concentration) + light/ultrasound treatment,\nsignificantly upon light and ultrasound exposure (Figure S30). HeLa cells\u2019 nuclei showed blue emission with Hoechst and red\nThe computed energy difference between the final and initial fluorescence with PI, indicating toward apoptotic cell death\nstates of the CO release is shown in Figure S31. For Re3, the mechanism for both photoactivated cancer therapy and SDT\n\u0394G between these two states in S0 PES is 5.99 eV, whereas the remaining inactive without drug stimuli (Figures S32\u2212S34).74\n\u0394G between these two states in T1 PES is 4.16 eV at the Moreover, the nuclei, as well as the cells, seemed to be\n\u03c9B97X-D/def2-TZVP//\u03c9B97X-D/def2-SVP level of theory. rounded up. The condensed nuclei were noted to be bright.\nThese energy differences show that the reaction is endothermic Therefore, it can be implied that Re2 and Re3 induced\n 6543 https://doi.org/10.1021/acs.jmedchem.3c02485\n J. Med. Chem. 2024, 67, 6537\u22126548\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\napoptosis in HeLa cells under light or ultrasound exposure. A calcd, 508.9679; found, 508.9658. UV\u2212visible spectral data were\nsimilar observation was also found with Re(I) complexes recorded in 10% aq. DMSO where \u03bbmax = 372 nm (\u03b5 = 2.08 \u00d7 104\nreported by Wilson and co-workers, showing the light- M\u22121 cm\u22121). 1H NMR (500 MHz, DMSO-d6): \u03b4 9.45 (dd, J = 5.0, 1.4\ntriggered apoptosis as the cell death mechanism.26 In contrast, Hz, 2H), 8.99 (dd, J = 8.3, 1.4 Hz, 2H), 8.35 (s, 2H), 8.13 (dd, J =\n 8.2, 5.0 Hz, 2H). IR (cm\u22121): 2016 (s, sh, and assym CO), 1935 (s, sh,\na cyanine-based Re(I) tricarbonyl induced ferroptosis under and assym CO), 1890 (s, sh, and assym CO), 1423 (m and sh), 851\nultrasound exposure.36,59 (s and sh) [m, medium; s, strong; br, broad; sh, sharp; assym,\n\n\u25a0 CONCLUSIONS\nIn summary, we reported the synthesis and characterization of\n asymmetric].\n Complex Re2. Orange solid, 82% yield. C15H7ClN3O5Re (MW =\n 530.89 g/mol) calcd: C, 33.94; H, 1.33; N, 7.92. Found: C, 34.16; H,\nthree Re(I)-tricarbonyl complexes (Re1\u2212Re3). The crystal 1.36; N, 7.83. Re2 has >95% purity. HRMS (m/z) [M + Na]+: calcd,\n 553.9529; found, 553.9536. [M + CH3CN\u2212Cl]+: calcd, 537.0209;\nstructure of complex Re2 confirmed the distorted octahedral found, 537.0206. [M \u2212 Cl]+: calcd, 495.9943; found, 495.9937. UV\u2212\ngeometry of the complexes. The emission properties of Re2 visible spectral data were recorded in 10% aq. DMSO where \u03bbmax =\nandRe3 were used to determine their internalization inside 328 (\u03b5 = 3.85 \u00d7 104 M\u22121 cm\u22121), 382 (\u03b5 = 2.59 \u00d7 104 M\u22121 cm\u22121). 1H\ncancer cells. The TD-DFT data indicated that the energy gap NMR (500 MHz, DMSO-d6): \u03b4 9.60 (dd, J = 11.9, 5.1 Hz, 2H), 9.41\nbetween the triplet excited state and the ground state (d, J = 2.6 Hz, 1H), 9.33 (dd, J = 8.6, 2.1 Hz, 1H), 9.21 (dt, J = 8.1,\n(\u0394ES0\u2212T1) is more than 0.98 eV, which is required to produce\n d d\n 1.8 Hz, 1H), 8.30\u22128.19 (m, 2H). IR (cm\u22121): 2020 (s, sh, and assym\n1\n O2. The visible band at 400\u2212500 nm for Re3 was helpful in CO), 1918 (s, sh, and assym CO), 1893 (s, sh, and assym CO), 1537\n (m and sh), 1418 (m and sh), 1329 (b and sh).\nobtaining visible light-assisted 1O2 generation, CO release, and Complex Re3. Reddish solid, 64% yield. C15H9ClN3O3Re (MW =\nanticancer responses. Re2 and Re3 were nontoxic (IC50 > 50 500.91 g/mol) calcd: C, 35.97; H, 1.81; N, 8.39. Found: C, 36.21; H,\n\u03bcM) in the absence of light to HeLa cells but potentially 1.89; N, 8.31. Re3 has >95% purity. HRMS (m/z) [M + Na]+: calcd,\ninduced apoptosis (IC50 = ca. 2.0 \u03bcM) on visible-light 523.9788; found, 523.9783. [M \u2212 Cl]+: calcd, 466.0201; found,\nexposure. Re2 did not display efficient cytotoxic effects upon 466.0196. UV\u2212visible spectral data were recorded in 10% aq. DMSO\nultrasound exposure, while Re3 still displayed cytotoxicity on where \u03bbmax = 367 nm (\u03b5 = 4.37 \u00d7 104 M\u22121 cm\u22121), 450 nm (\u03b5 = 0.95 \u00d7\nultrasound exposure (IC50 = 5.0 \u03bcM). Re3 has a high 104 M\u22121 cm\u22121). 1H NMR (500 MHz, DMSO-d6): \u03b4 (ppm) 9.39 (dd, J\nphotocytotoxicity index (>25), suggesting selective anticancer = 5.1, 1.2 Hz, 1H), 9.13 (dd, J = 8.5, 1.3 Hz, 1H), 8.94 (dd, J = 4.9,\n 1.3 Hz, 1H), 8.50 (dd, J = 8.4, 1.2 Hz, 1H), 8.07 (dd, J = 8.5, 5.1 Hz,\nactivity efficacy under visible-light exposure. Importantly, 1H), 7.80 (dd, J = 8.4, 4.9 Hz, 1H), 7.07 (s, 1H), 6.90 (s, 2H). IR\nagainst normal HEK cells, Re2 and Re3 did not produce any (cm\u22121): 2021 (s, sh, and assym CO), 1895 (s, sh, and assym CO),\nnoticeable toxicity, indicating their tumor-targeting anticancer 1634 (m and sh), 1432 (m and sh), 1313 (b and sh).\npotential. Interestingly, Re3 was not only able to generate ROS DFT Calculation. The DFT and TD-DFT calculations were\nbut also induced NADH to NAD+ photo-oxidation. To the carried out on neutral Re complexes with the Gaussian 16 revision\nbest of our knowledge, NADH photo-oxidation has been A.03 quantum chemistry package. For all calculations, the \u03c9B97X-D/\nachieved with the Re(I) complex for the first time here. Def2-TZVP level of theory was used using the CPCM solvation\nMoreover, Re3 released CO upon ultrasound and light model in DMSO. The ground-state (S0) geometry of the complexes\nexposure. The CO release profile of Re3 indicated the was carried out using restricted DFT, the S1 geometry using TD-DFT\n and the first excited triplet-state (T1) geometry using unrestricted\nnecessity of light/ultrasound as a trigger. The released CO DFT. The optimized S0 and T1 structures were confirmed to local\npossibly contributed to the overall cytotoxicity of Re3. minima at the same computational level. Natural transition orbitals\nConsidering the above-mentioned mechanistic evidence, this (NTOs) and frontier molecular orbitals (FMOs) were generated at\nstudy indicates the ability of Re3 to act as multitargeting and the same level of theory. The redox as well as the photoredox\nmultifunctional (ROS generation, NADH photo-oxidation, and potentials of Re1\u2212Re3 were evaluated using the Born\u2212Haber cycle.\nCO release) anticancer agents under the influence of light or The standard potentials at 293 K were determined from the solvated\nultrasound. Even though Re3 presented better activity under free energies using\nvisible light than ultrasound, it can still be useful for superficial Gred(sol)\n 0\ncarcinomas only due to visible light\u2019s poor tissue penetration Eabs = 0.03766\n nF\nability. However, the activation of Re3 with ultrasound might\nbe used to treat buried tumors due to ultrasound\u2019s deep tissue- where n = 1 (no. of free electrons), \u0394Gred(sol) is the Gibbs free\npenetration ability. energy, and F is the Faraday constant for the solvated reduction half-\n reaction, and they were calculated vs Ag/AgCl.\n\n\u25a0 EXPERIMENTAL SECTION\n General Synthetic Procedure. 5-nitro-1,10-phenanthroline\n NADH Oxidation. Reactions between Re3 (10 \u03bcM) and NADH\n (140 \u03bcM) in DMSO-PBS (2:98 v/v) solution were monitored by\n UV\u2212vis spectroscopy at ambient temperature in the dark or light\n(phen-NO2) and 5-amino-1,10-phenanthroline (phen-NO2) were irradiation.65 The turnover number (TON) and turnover frequency\nsynthesized according to the method described in the literature (TOF) of catalysis were calculated using the following equations\n(Scheme S1).27 1.0 equiv of phen-based ligands [1,10-phenanthroline\n(in Re1), 5-nitro-1,10-phenanthroline (in Re2), and 5-amino-1,10- [NAD+] = [Abs(339 nm)i Abs(339 nm)f ]/Abs(339 nm)i\nphenanthroline (in Re3)] and 1.0 equiv of Re(CO)5Cl were dissolved\n \u00d7 [NADH]\nin 15\u221220 mL of toluene (Scheme S1).27 The reaction mixture was\nrefluxed for 2 h under inert conditions. After 1\u22121.5 h, precipitation\n TON = [NAD+]/[Re3]\nstarted. The precipitated complexes were filtered, washed with hexane\nand Et2O, and dried in a vacuum over P4O10. The complexes were TOF = TON/time (h)\nrecrystallized from a DCM\u2212ethanol mixture. The purity of complexes\nwas determined by elemental analysis and NMR data, and the result Detection of H2O2 Generation. During the reaction of Re3 (10\nindicated >95% purity. \u03bcM) with NADH (140 \u03bcM) in the DMSO-PBS solution (2:98 v/v) at\n Complex Re1. Yellow powder, 85% yield. C15H8ClN2O3Re (MW = ambient temperature in the dark or after light (400\u2212700 nm, 5 J\n485.89 g/mol) calcd: C, 37.08; H, 1.66; N, 5.77. Found: C, 37.21; H, cm\u22122) exposure for 16 min, H2O2 was detected by Quantofix peroxide\n1.73; N, 5.84. Re1 has >95% purity. HRMS (m/z for [M + Na]+): test sticks.65 The H2O2 generation level in the solution can be\n\n 6544 https://doi.org/10.1021/acs.jmedchem.3c02485\n J. Med. Chem. 2024, 67, 6537\u22126548\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\ncorrelated to the color change from white to blue (of the test sticks), microscope in phase contrast, red, and blue channels at 400\u00d7\nindicating 0\u221225 mg/L amount of H2O2. magnification.\n 1\n O2 Generation. The 1O2 generation was measured using the\nDPA probe upon light or ultrasound irradiation.36 In brief, a DMSO/\nH2O (2:98 v/v) solution containing 10 \u03bcM of Re1\u2212Re3 and 0.3 \u03bcg/\nmL of DPA was monitored by UV\u2212vis spectroscopy on different\n \u25a0 ASSOCIATED CONTENT\n * Supporting Information\n s\u0131\n\nintervals of ultrasound (1.5 W cm\u22122) or light (400\u2212700 nm, 5 J cm\u22122) The Supporting Information is available free of charge at\nexposure. The absorbance of the ca. 378 nm peak was monitored for https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c02485.\n1\n O2 generation. The 1O2 quantum yields (\u03a6\u0394) for Re1\u2212Re3 were\ndetermined by following equation Crystal data parameters, FMO energies, singlet\u2212singlet/\n triplet transition energies, adiabatic singlet\u2212triplet\n S\n = R\n \u00d7 (MS/MR ) splitting energies, computed redox potentials, character-\n ization data (1H and 13C NMR, HRMS, FT-IR), unit cell\nwhere \u201cM\u201d denotes the linear fit slope of the DPA-based peak at 378 packing, FMOs, NTOs, HOMO\u2212LUMO energy gap,\nnm vs the time interval. \u201cS\u201d denotes the samples, and \u201cR\u201d denotes the optimized geometries in S1 and T1, SOMOs, 1O2\nstandard ([Ru(bpy)3]Cl2). generation, OH\u2022 generation, in-cell ROS generation,\n Detection of OH\u2022 Generation. The production of hydroxyl NADH oxidation, and confocal images for cell death\nradical (OH\u2022) in solution was detected using MB as the OH\u2022 probe.36 mechanism (PDF)\nThe reaction between 10 \u03bcM Re1\u2212Re3 and 1 \u03bcg/mL MB in DMSO/\nH2O (2:98 v/v) solution was monitored by UV\u2212vis spectroscopy Crystallographic data for complex Re1\u2212Re3 (CIF)\nafter different intervals of ultrasound (1.5 W cm\u22122) and visible-light Check CIF/PLATON report (PDF)\n(400\u2212700 nm, 5 J cm\u22122) irradiation.\n Cytotoxicity Assay (Both Light and Ultrasound). The Molecular formula strings of Re1\u2212Re3 (CSV)\ncytotoxicity assay of Re1\u2212Re3 has been performed against HeLa\ncells (cervical carcinoma), MCF-7 cells (mammalian breast\nadenocarcinoma), and HEK cells (normal cell line). In brief, 10,000\ncells/well were seeded in 3 different (one for light exposure, second\n \u25a0 AUTHOR INFORMATION\n Corresponding Authors\none for dark conditions, and third one for ultrasound exposure) 96- Samya Banerjee \u2212 Department of Chemistry, Indian Institute\nwell cell culture plates and incubated overnight for adherence. After of Technology (BHU), Varanasi, Uttar Pradesh 221005,\nincubation, the cells were treated at various concentrations of each India; orcid.org/0000-0003-4393-4447;\ndrug (500 nM, 750 nM, 1 \u03bcM, 5 \u03bcM, 10 \u03bcM, 20 \u03bcM, and 50 \u03bcM) and\nkept for a further 6 h incubation. Thereafter, the drug-containing Email: samya.chy@itbhu.ac.in\nmedium was discarded and 100 \u03bcL PBS was added into each well of Biplob Koch \u2212 Department of Zoology, Institute of Science,\nall treated 96-well plates. One of those plates was exposed to light Banaras Hindu University, Varanasi, Uttar Pradesh 221005,\n(400\u2212700 nm, 5 J cm\u22122) for 30 min, and subsequently, one was kept India; Email: biplob@bhu.ac.in\nin a dark condition and the third one was irradiated with ultrasound Tumpa Sadhukhan \u2212 Department of Chemistry, SRM\n(for 10 min). After 30 min, PBS was removed from both plates, and a Institute of Science and Technology, Kattankulathur, Tamil\nfresh complete medium was added and incubated for another 18 h. Nadu 603203, India; Email: tumpas@srmist.edu.in\nFinally, the medium was discarded, and the fresh MTT-containing\nmedium was added to each well. After 2 h of incubation, the MTT- Authors\ncontaining medium was removed, and 100 \u03bcL DMSO was added into Rajesh Kushwaha \u2212 Department of Chemistry, Indian\neach well and further incubated for 0.5 h. Thereafter, absorbance was Institute of Technology (BHU), Varanasi, Uttar Pradesh\nrecorded in a multiplate reader at 570 nm.\n ROS Generation (Both Light and Ultrasound). The assay was\n 221005, India\nperformed by seeding 0.5 \u00d7 105 HeLa cells/well in 12-well cell culture Virendra Singh \u2212 Department of Zoology, Institute of Science,\nplates followed by treatments with compounds Re2 and Re3 at their Banaras Hindu University, Varanasi, Uttar Pradesh 221005,\nrespective IC50 value concentration and incubated for 6 h. A fresh PBS India\nwas added to each well after discarding the drug-containing medium, Silda Peters \u2212 Department of Chemistry, SRM Institute of\nand one plate was exposed to light, another one was irradiated with Science and Technology, Kattankulathur, Tamil Nadu\nultrasound, and subsequently, one was kept in dark condition. After 603203, India\nlight and ultrasound irradiation, PBS was removed, and fresh DMEM Ashish Kumar Yadav \u2212 Department of Chemistry, Indian\nwas added to each plate and incubated for another 18 h. The cells Institute of Technology (BHU), Varanasi, Uttar Pradesh\nunderwent a PBS wash, followed by addition of 10 \u03bcM DCFDA,\nfollowed by incubation at 37 \u00b0C for 0.5 h. Finally, images were\n 221005, India\nphotographed in a fluorescent microscope under green channels and Complete contact information is available at:\nphase contrast at 100\u00d7 magnification. https://pubs.acs.org/10.1021/acs.jmedchem.3c02485\n Apoptosis Study by Hoechst/PI Dual Staining (Both Light\nand Ultrasound). To investigate the nuclear morphology changes\n Author Contributions\nafter Re2 and Re3 treatments, we performed Hoechst 33342/PI dual\nstaining. For this, 50,000 HeLa cells/well were seeded in 12-well R.K. and A.K.Y. synthesized and characterized the complexes.\nplates supplemented with complete DMEM followed by treatments V.S. performed the biological assays. R.K. performed ROS\nwith Re2 and Re3 at their respective IC50 value concentration and generation and NADH photo-oxidation studies. S.P., R.K., and\nincubated for 6 h. Then, the drug-containing medium was discarded, T.S. performed the TD-DFT calculations. S.B., B.K., and T.S.\nfresh PBS was added to each well, and one plate was exposed to light designed the studies and formulated the concept and overall\n(400\u2212700 nm, 5 J cm\u22122), another one was irradiated with ultrasound, project. The manuscript was written through the contributions\nand one was kept in dark condition. After light and ultrasound of all authors. All authors have approved the final version of the\nirradiation, PBS was removed, and fresh DMEM was added to each manuscript.\nplate and incubated for another 18 h at 37 \u00b0C in a humidified 5% CO2\nincubator. Finally, staining with 10 \u03bcg/mL PI and 10 \u03bcg/mL Hoechst Notes\n33342 was done, and images were captured in a fluorescent The authors declare no competing financial interest.\n 6545 https://doi.org/10.1021/acs.jmedchem.3c02485\n J. Med. Chem. 2024, 67, 6537\u22126548\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n\n\u25a0 ACKNOWLEDGMENTS\nWe thank SERB (SRG/2022/000030), the Government of\n (14) Imberti, C.; Zhang, P.; Huang, H.; Sadler, P. J. New designs for\n phototherapeutic transition metal complexes. Angew. Chem. Int. Ed.\n 2020, 59, 61\u221273.\nIndia, and BRNS (54/14/08/2022-BRNS-R) for financial (15) Mandal, A. A.; Kushwaha, R.; Yadav, A. K.; Banerjee, S. Metal\nsupport. R.K. and A.K.Y. thank the Ministry of Education, the Complexes for Cancer Sonodynamic Therapy. ChemBioChem 2023,\nGovernment of India, for the Prime Minister\u2019s Research 24, No. e202200597.\nFellowship. B.K. acknowledges the Indian Council of Medical (16) Bonnet, S. Ruthenium-Based Photoactivated Chemotherapy. J.\nResearch, New Delhi, India, for providing financial support Am. Chem. Soc. 2023, 145, 23397\u221223415.\n(no. 5/13/36/2022-NCD-111) and Banaras Hindu University (17) Liang, C.; Xie, J.; Luo, S.; Huang, C.; Zhang, Q.; Huang, H.;\n Zhang, P. A highly potent ruthenium (II)-sonosensitizer and\nfor providing funding under the IoE scheme (file no. R/Dev/ sonocatalyst for in vivo sonotherapy. Nat. Commun. 2021, 12, 5001.\nD/IoE/Incentive/2021-22/32449). We thank the high-per- (18) Banerjee, S.; Chakravarty, A. R. Metal Complexes of Curcumin\nformance computing facilities at IIT BHU and SRMIST. 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